JP2020501138A - Apparatus for demonstrating the effect of active ingredients on nematodes and other organisms in aqueous tests - Google Patents

Abstract

The present invention relates to a device 1 for recording an entire image of a cell culture plate comprising one or more cavities, the device 1 being a holder 40 for receiving a cell culture plate 50, wherein the cell culture plate comprises: A holder 40 having a bottom surface 51, a top surface 52, and a plurality of side surfaces extending between the bottom surface and the top surface, and functions to record an image of the bottom surface of the cell culture plate within a predetermined depth of field. The lighting device includes a camera 20 and a lighting device 30 for lighting the cell culture plate, and the lighting device includes a plurality of light sources. At the use position of the cell culture plate, the light of the first, second, third, and fourth light sources passes through the entire length of the first, second, third, and fourth sides from the outside, and the cell culture plate to go into. The intensity of the emitted light, the vertical height of the emitted light with respect to the cell culture plate, the horizontal distance of the light source from each side, and the angle of inclination of the emitted light with respect to the axis are adjusted by the adjusting means of the lighting equipment. Individually adjustable for the light source.

Description

  The present invention relates to a device for demonstrating the effect of an active ingredient on nematodes and other organisms in aqueous tests. Furthermore, the invention relates to a method for adjusting the lighting equipment of the device.

  These are agricultural pests, as many species of nematodes (roundworms) can significantly impair plant metabolism as a result of their entry into the root system. Various chemicals, so-called nematicides, have already been developed against attack by nematodes. However, there is a high demand to identify additional active ingredients that can effectively control nematodes.

  WO 2016/116291 (A1) discloses a device for demonstrating the effect of an active ingredient on nematodes, whereby a very large number of active ingredients can be tested in a short period of time. At this time, individual wells of a cell culture plate filled with nematodes and various active ingredients are simultaneously examined. The cell culture plate held by the holder of the device has a bottom surface, a top surface and four sides extending between the bottom and top surfaces of the cell culture plate. The device is equipped with a camera that functions to record images of the bottom of the cell culture plate, and thus of all wells at the same time. The lighting equipment of the device comprises two opposing light sources that illuminate the cell culture plate.

  The application of the device from WO 2016/116291 (A1) has been found to reach its limits when examining very small roundworms such as canine filamentous worms. The resolution of the device can be increased, but in principle, by choosing a correspondingly higher resolution camera, the non-uniformity of the illumination of the cell culture plate can no longer be satisfied by the software at higher resolutions. It turns out that it cannot be corrected to the extent possible. As a result, a reliable and robust demonstration of the effect of the active ingredient is no longer possible for very small organisms.

  Accordingly, the present invention provides an apparatus for demonstrating the effect of an active ingredient on nematodes and other organisms, even in the case of very small organisms such as canine filamentous worms, by reliable evidence of the effect of the active ingredient. Based on the purpose of

  The object of the invention is achieved by using a device according to claim 1. Exemplary embodiments of the invention can be gathered from the dependent claims relating to claim 1.

  The device according to the invention comprises, in each case, the light of the first light source passing through the first side from the outside and entering the cell culture plate, the second light source passing through the second side from the outside and entering the cell culture plate. The light is identified by the light of the third light source passing through the third side from the outside and entering the cell culture plate, and the light of the fourth light source passing through the fourth side from the outside and entering the cell culture plate. As a result, light enters the cell culture plate from all sides. In principle, a uniform illumination of the cell culture plate in the sense of the present invention is possible as a result of this measure. As a result, even for very small organisms, the effect of the active ingredient can be examined in a cell culture plate with a correspondingly powerful camera. Consequently, robust results can be obtained for resolutions better than 30 μm. In one exemplary embodiment, the resolution is better than 20 μm, for example, 15 μm.

  The light intensity can be set individually for the first light source. This also applies to the second, third and fourth light sources. As an example, it is consequently possible to set the light intensity of the first light source independently of the light intensity of the second light source or another light source. After all, the four light sources can thus emit light at different light intensities to the cell culture plate in each case.

  In the position of use of the cell culture plate (in this case, the cell culture plate extends in a horizontal plane), the light of the first light source is Penetrating the first side, substantially horizontally, into the cell culture plate. Consequently, possible polarizing mirrors or other light polarizations can be omitted.

  The description for the first light source should likewise apply to the other light sources. Thus, for example, the second light source can also be arranged laterally adjacent to the second side surface. The features relating to the first light source and the features described below can be performed in one of the remaining light sources, in some of the remaining light sources, or else in all of the remaining light sources.

  The present invention contemplates in each case a square cell culture plate having a square basic shape with two parallel sides. As an example, the ratio of the long side (long side) to the short side (short side) can be 3 to 2. Usually, the wells are arranged in a plurality of rows arranged in parallel with each other. By way of example, a cell culture plate can have 8 rows with 12 wells in each case. Typically, square microplates with 6, 12, 24, 96 or 384 wells are used according to ANSI standards.

  The present invention is based on the discovery that the good illumination of all wells of a cell culture plate required when examining very small organisms can be performed by lighting equipment when light enters the cell culture plate from all sides. Based. However, for hexagonal cell culture plates whose corresponding basic shape is rarely found at present, all six sides would have to be illuminated according to the invention. For triangular cell culture plates, the use of only three light sources may be sufficient.

  Typically, the sides extend between the top and bottom surfaces of the cell culture plate, having light transmissive walls.

  Usually, the first light source is spread over the entire large side, so that a uniform illumination of the first side is possible.

  In one exemplary embodiment, the vertical height with respect to the cell culture plate, the angle of inclination with respect to its own axis and / or the horizontal distance from the first side are individually set for the first light source by the adjusting means of the lighting device. can do.

  Illumination of the cell culture plate can be optimized as a result of the flexible and individually adjustable settings of each light source. At this time, when viewed spatially, each light source can be adjusted in three dimensions (height, horizontal distance, tilt angle). In addition, the fourth dimension, specifically the light intensity, can be set. The precise individual setting of each side light source is very important for the quality of the measurement, even for contrasting cell culture plates with a square basic shape.

  Preferably, each light source is connected to a controller. Particularly preferably, each light source is assembled on a holder connected to the controller or another controller.

  The vertical height of the light beam emanating from the first light source may be 2-6 mm, preferably 3-5 mm. In the case of a lateral arrangement of the first light source, this light beam without the oblique angle passes through the first side, i.e. horizontally into the cell culture plate between the top and bottom surfaces.

  Preferably, the aperture angle of the light beam is very small, for example, so that the cover film covering the individual wells of the cell culture plate is not illuminated and cannot result in interference effects. However, for example, only a small vertical portion of the cell culture plate is imaged in focus by the camera, so that the plane of the cell culture plate where the cover film is located is no longer in focus, and larger values of the aperture angle are acceptable. can do. In this embodiment, all incident light is maintained so that the cell culture plate is illuminated only from the bottom to the depth of field defined by the lens. The cover or protective film is preferably not illuminated to avoid light reflection.

  Preferably, the controller is configured to automatically execute the lighting device adjustment method.

  The first light source may comprise a number of light emitting diodes (LEDs) arranged next to each other. In order to emit uniform light over the effective length of the first light source, the first light source can have a Fresnel lens arrangement. As a result of this Fresnel lens arrangement, the first light source will eventually emit a light beam whose intensity is constant when viewed along the axis of the light source.

  A holder for the cell culture plate (also called an MTP holder) receives a plurality of receiving corners, a first receiving corner that functions to receive one end of the first side, and an opposite end of the first side. It has a functioning second receiving corner. At this time, the distance between the first receiving corner and the second receiving corner is preferably longer than the length of a row of wells extending along the first side surface. The light of the first light source eventually enters the unobstructed cell culture plate over the entire length of the row of wells. Preferably, a light source of the first light source is disposed between the first light source and the first side of the cell culture plate to promote unobstructed light into the cell culture plate and to enter the cell culture plate without being shaded. Materialize the holder so that there are no other obstacles.

  In order to eliminate or at least largely eliminate the perspective aberration of the image at the contours of the cell culture plate, the camera may be equipped with a telecentric lens for precision measurement (eg, from Sill Optics).

  Usually, a digital camera equipped with an image sensor is used. Preferably, the bottom of the cell culture plate is adjusted with respect to the image sensor surface (also referred to as chip tilt), and the cell culture plate is adjusted with maximum precision parallel to the image sensor (preferably, surface and side). Next, rotate the cell culture plate with respect to the image sensor. For adjustment purposes, the MTP holder is usually precisely adjustable in all directions, for example by tightening to a defined strength.

  As a result of applying the telecentric lens in combination with an adjustable cell culture plate assembled on an MTP holder, it was possible to perform <± 2% distortion, ie, a flat image recording without software correction. . Furthermore, a shallow depth of field can be set by the aperture of the telecentric lens, so that, for example, the interference effects on the wells caused by the aforementioned cover film can be reduced.

  The device according to the invention facilitates image analysis of the complete plate surface and is suitable, for example, for cell culture plates with up to 384 wells containing microorganisms such as canine filamentous worms (remodeling limit approx. 15 μm). Obviously, a full-length image of a cell culture plate containing organisms or particles suspended in a solution can generally be recorded with the device according to the invention and processed by image analysis.

  During operation of the device, the camera records a plurality of digital images at intervals of, for example, 1-5 seconds, for example, over the entire bottom surface of a cell culture plate with 96 wells. For the purpose of evaluating these images based on pixel analysis, certain characteristics can be established for each individual well, for example with respect to the average speed at which the canine filamentous worms in this well move. At this time, based on the principle, the average speed in the wells containing the active ingredient is lower than in the wells without the active ingredient. Consequently, the effectiveness of the active ingredient can be estimated based on the average speed. In this connection, reference is made to WO 2016/116291 (A1), in which a corresponding method for establishing the effect of the active ingredient is described in detail, on which the device according to the invention is operated. It is possible.

  The same with respect to the movement of canine filamentous worms in the case of cell culture plates illuminated uniformly throughout and in the case of wells filled to the same extent in each case (e.g. without active ingredient in each case) The average rate should theoretically occur for each well if necessary to eliminate the current biological variability. In contrast, if the wells are under-illuminated, the results deviate significantly, despite the same fill. Thus, in the case of a cell culture plate with wells filled with different active ingredients illuminated in this way, references to individual active ingredients will not be robust. However, the lighting equipment of the device according to the invention has the effect of illumination differences that cannot be virtually eliminated at all, even in very small canine filamentous worms (i.e. in the corresponding high resolution). It allows to keep as small as possible a robust statement about the effect.

  A further object was to provide a method for adjusting the lighting equipment of the device in various embodiments thereof. This object is achieved by a feature combination according to claim 12. Exemplary embodiments of the method according to the invention can be gathered from the dependent claims relating to claim 12.

  For each of the light sources, the method according to the invention provides a method of obtaining the illumination of the individual wells of the cell culture plate that is as uniform as possible, in relation to the height of the cell culture plate, the angle of inclination, the horizontal to the adjacent side. Provides distance and individually set light intensity.

  In a systematic way, the individual parameters (height, distance, tilt angle, light intensity) can be set repeatedly for each light source, and using these setting parameters, It is possible to make the above demonstration of the average speed for the filled wells. This method can be repeated as often as desired. The less the proven results of the average velocity in the method deviate, the less the effect of uneven illumination of the cell culture plate on the test results. Finally, if the results deviate only by an amount that can be attributed to biological variation, the adjustment method can be completed.

  The method of adjusting the lighting device can be to place the sheet on an unfilled cell culture plate that is in a flat plate on the cell culture plate (e.g., in a holder with an additional empty cell culture plate inserted). Adjustments to make or improve may be provided based on the camera and image evaluation used to create the image of (a). As an example, a histogram of light intensity can be obtained from the image, and adjustments can be made based on this histogram. It will mean that the histogram consists simply of one bar with this one light intensity, in each case the associated histogram is the same for all sub-regions (these could also be individual pixels of the digital image) Provides uniform illumination of the sheet when having light intensity.

  In one exemplary embodiment, the lighting equipment is first adjusted by the results of the cell culture plate with wells of equivalently filled wells, as well as by the sheet. The (first) adjustment based on the equally filled cell culture plate and the (post) adjustment by the sheet can be performed multiple times continuously and / or in reverse order.

  Furthermore, the light intensity of the individual light sources may be reduced by the same magnitude in each case, or by the average light intensity of the image of the monochromatic sheet of paper (for example, an average of 128 if the light intensity can be assumed to be a value between 0 and 256). At the end of the adjustment in such a way that a predetermined value for (value) is obtained. Then, during operation of the device according to the invention, each well of the cell culture plate is assigned a corresponding area on the paper sheet. The light intensity values for the pixels of the well as verified by the camera can then be normalized based on the light intensity of the corresponding area on the paper sheet. As an example, if rather the lighter area of the paper sheet is established for a well, the light intensity values established for this well are reduced by normalization.

The present invention should be described in more detail based on the exemplary embodiments shown in the figures. In the figure:
FIG. 1 schematically shows the structure of the device according to the invention;
FIG. 2 schematically shows a lighting device with a cell culture plate and four light sources; and FIG. 3 shows a cross-sectional view along the line III-III in FIG.

  FIG. 1 schematically shows a device for demonstrating the effect of an active ingredient on canine filamentous worms and other organisms in an aqueous test. The device, generally designated 1, comprises a housing 10 having an upper separator 11 and a center separator 12. Separators 11, 12 divide the interior surrounded by housing 10 into an upper region 13, a central region 14, and a bottom region 15. The camera 20 is assembled in the bottom area 15. The camera 20 comprises a telecentric lens 21 whose end 22 facing outwardly from the camera 20 projects into the central region 14 of the housing 10. The center separating plate 12 has a circular opening 16 through which the lens 21 passes. At this time, the frustoconical portion 23 of the lens 21 is supported on the circular contour of the opening 16.

  The lighting device 30 and the holder 40 of the cell culture plate 50 are housed in the upper region 13 of the housing 10. The lighting device 30, the holder 40 and the cell culture plate 50 are further described in more detail below based on FIGS.

  In addition to the camera 20, the bottom area 15 of the housing 10 also houses a controller 60 for the camera 20 and the lighting device 30. At this time, the controller 60 may be a separate controller for the camera 20 and the lighting device 30. The controller 60 can be connected to a computer.

  With the device 1 according to the invention, it is possible to obtain an image of the bottom surface 51 of the cell culture plate 50. Therefore, the glass panel 17 is incorporated in the separation plate 11.

  FIG. 2 shows the lighting device including the lighting device 30, the first light source 31, the second light source 32, the third light source 33, and the fourth light source 34. The first light source 31 includes a plurality of light emitting diodes (not shown) that are arranged adjacent to each other in a row and emit the band light beam 35 over substantially the entire length of the first light source 31. The rays are illustrated by parallel arrows 35. Over substantially its entire length, the remaining light sources 32, 33, 34 also each emit a band of light in the direction of the cell culture plate 50. However, the corresponding arrows labeling each ray are not shown in FIG. 2 for clarity.

  In addition to the bottom surface 51 already mentioned above, the cell culture plate 50 comprises a top surface 52 and a first side 53, a second side 54, a third side 55 and a fourth side 56. Due to the square basic shape of the cell culture plate 50, the opposite sides 53, 54 extend perpendicular to the other pair of sides 55, 56 extending in parallel.

  The cell culture plate 50 comprises a very large number of wells 57 arranged in 8 rows, each containing 12 wells. As can be gathered from FIG. 3, each individual well 57 has a U-shape in longitudinal section. At this time, each well 57 can be filled with an aqueous solution from above, i.e., from the top surface 52, a canine filamentous worm, and the active ingredient in said aqueous solution. Once filling is complete, the wells can be covered by a cover sheet.

  2 and 3, the individual light sources 31, 32, 33, 34 each emit light into the cell culture plate 50 through the sides 53, 54, 55, 56. . Therefore, light from the first light source passes through the first side surface 53 from the outside and passes through the cell culture plate 50. The same applies to light sources 32, 33, 34 as well. In order to achieve the best possible illumination of the individual wells 57 of the cell culture plate 50, the position of the light source 31 can be modified relative to the cell culture plate 50.

  The double arrow 36 indicates that the distance between the first light source 31 and the first side surface 53 of the cell culture plate 50 can be increased or decreased. A double arrow 37 (see FIG. 3) indicates that the first light source 31 can be moved up and down with respect to the cell culture plate 50. Further, the first light source 31 can be tilted with respect to the axis 38 extending parallel to the first side 53 of the cell culture plate 50. A corresponding adjustment with respect to the axis 38 or an axis extending parallel thereto is indicated by a curved arrow 39. Further, the light intensity of the first light source 31 can be set by the controller 60. The horizontal distance from each side, the vertical position, the tilt angle and the light intensity of each light source can be individually set by the corresponding setting parameters of the remaining light sources 32, 33, 34. As a result, for example, the lighting device is adjusted such that the light intensity of the first light source 31 is not only deviated from the light intensity of the light sources 33 and 34 arranged in the lateral direction thereof, but also from the light intensity of the light source 32 on the opposite side. It is possible to

  The holder 40 includes a first receiving corner 41, a second receiving corner 42, a third receiving corner 43, and a fourth receiving corner 44. Preferably, three of the four receiving corners 41, 42, 43, 44 are embodied as stationary stops, and the remaining fourth receiving corner is embodied as a spring-loaded stop. For example, the distance between the first receiving corner and the second receiving corner is dimensioned such that the light ray 35 of the first light source 31 hits without obstructing the area of the first side surface where the well 57 is located. In other words, the distance between the receiving corners 41, 42 is longer than the length of the first row of wells extending along the first side 53.

  Due to the different lengths of the longer sides 55, 56 and the shorter sides 53, 54, one light source 33, 34 and the other light source 31, 32 have different lengths. In this case, the width of the light beam from the light source is always longer than the length of the area of the relevant side where the well is located.

  The entire area of the side of the cell culture plate 50 where the wells 57 are located is affected by the light from the light source as a result of the lighting device 30 and the special arrangement of the individual receiving corners of the holder 40. As a result of the individual adjustment of the individual light sources, the luminaire 30 can be adjusted to achieve almost optimal illumination of the well 57. Preferred monitoring criteria for illumination that is as optimal as possible have already been described above. Well-specific results in cell culture plates whose individual wells are in each case filled with the same active ingredient do not deviate in each case against the efficacy of the same active ingredient or because of biological organisms If it has a standard deviation approximately equivalent to the estimated standard deviation, an optimal illumination can be assumed.

DESCRIPTION OF SYMBOLS 1 Device 10 Housing 11 Upper separating plate 12 Central separating plate 13 Upper region 14 Central region 15 Bottom region 16 Opening 17 Glass plate 20 Camera 21 Lens 22 End 23 Frustoconical region 30 Lighting equipment 31 First light source 32 Second light source 33 third light source 34 fourth light source 35 arrow (light ray)
36 Double arrow 37 Double arrow 38 Arrow 40 Holder 41 First receiving corner 42 Second receiving corner 43 Third receiving corner 44 Fourth receiving corner 50 Cell culture plate 51 Bottom 52 Top 53 First side 54 Second side 55 Third side 56 Fourth side 57 Well 60 Controller

Claims (14)

An apparatus (1) for recording an entire image of a cell culture plate having one or more wells,
A holder (40) for receiving said cell culture plate (50) with wells (57), said cell culture plate (50) comprising a bottom surface (51), a top surface (52) and said bottom surface (52); 51) a holder (40) comprising a plurality of side surfaces extending between 51) and said top surface (52);
A camera (20) operative to record an image of the bottom surface (51) of the cell culture plate (50) within a predetermined depth of field;
-An illumination device (30) for illuminating the cell culture plate (50);
With
The lighting device (30) includes a plurality of light sources for emitting light rays,
At the use position of the cell culture plate (50),
The light of the first light source (31) penetrates the entire length of the first side (53) from outside and enters the cell culture plate (50);
The light of the second light source (32) penetrates the entire length of the second side (54) from the outside and enters the cell culture plate (50);
The light of the third light source (33) penetrates the entire length of the third side (55) from the outside and enters the cell culture plate (50);
-The light of the fourth light source (34) passes through the entire length of the fourth side surface (56) from the outside and enters the cell culture plate (50);
The intensity of the emitted light beam, the vertical height of the emitted light beam with respect to the cell culture plate, the horizontal distance of the light source from each of the side surfaces, and the angle of inclination of the emitted light beam with respect to axis (38). Is individually adjustable for each light source (31-34) by adjusting means of said lighting equipment (30).   The device (1) according to claim 1, wherein the first light source (31) comprises a Fresnel lens arrangement for emitting uniform light over the effective length of the first light source (31).   3. The device according to claim 1, wherein each light source and / or a holder for fixing the light source is connected to one or more controls for adjusting the lighting device. Device (1).   4. The method according to claim 1, wherein the controller for adjusting the lighting device is configured to execute a method for adjusting the lighting device. 5. Apparatus (1).   Device (1) according to any of the preceding claims, wherein the first light source (31) is arranged laterally adjacent to the first side surface (53). .   The intensity of the emitted light beam, the vertical height of the emitted light beam with respect to the cell culture plate, the horizontal distance of the light source from each of the side surfaces, and the angle of inclination of the emitted light beam with respect to axis (38). Is set for each light source (31-34) such that said cell culture plate is illuminated from said bottom to said predetermined depth of field. Device (1) according to any one of the preceding claims.   Apparatus (1) according to any of the preceding claims, wherein the vertical height of the light beam emitted from the first light source (31) is between 2 and 6 mm.   The holder (40) includes a plurality of receiving corners, the first receiving corner (41) functions to receive one end of the first side surface (53), and the second receiving corner (42) includes It functions to receive the opposite end of the first side surface (53), and the distance between the first receiving corner (41) and the second receiving corner (42) is along the first side surface (53). Device (1) according to any of the preceding claims, characterized in that it is longer than the length of the rows or columns of the extending wells (57).   Device (1) according to any of the preceding claims, wherein the camera (20) comprises a telecentric lens (21).   The camera comprises an image sensor, and the cell culture plate (50) and / or the image sensor are positioned relative to each other in all directions such that the cell culture plate is adjusted in all directions parallel to the image sensor. Device (1) according to any of the preceding claims, wherein the device (1) is adjustable.   For the purpose of demonstrating the effect of the active ingredient on nematodes and other organisms in an aqueous test, the wells of the cell culture plate are filled with nematodes and other organisms in an aqueous solution, with or without the active ingredient. Device (1) according to any of the preceding claims, characterized in that:   A method for adjusting the lighting device (30) of the device (1) according to any one of claims 1 to 11, wherein a relative height of each light source with respect to the cell culture plate, an inclination angle of each light source. And the light intensity of each light source is individually set to obtain good illumination of the individual wells (57) of the cell culture plate (50) and uniform illumination of the cell culture plate. A method characterized by the following.   The camera (20) is used to create an image of a monochromatic sheet lying flat on the illuminated cell culture plate (50) and perform the adjustment based on the evaluation of the image. The method according to claim 12, wherein   The light intensity histogram is created from the image, and adjustments for the purpose of controlling the adjustment means of the lighting device (30) are performed based on the histogram until the histogram has a minimum width. The method according to claim 13, wherein:

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